Purpose: The goal of this study was to compare the efficacy of oral 25% dextrose treatment or/and pacifier for analgesia in healthy newborns during intramuscular injection of a hepatitis B vaccine. Methods: A prospective, randomized, partially blinded, clinical trial was performed in 132 healthy newborns. They were assigned randomly to 4 treatment groups: control group (2 mL distilled water), dextrose group (2 mL 25% dextrose), pacifier group, dextrose+pacifier group (pacifier coating with 25% dextrose) during intramuscular injection of hepatitis B vaccine. For all groups, Neonatal Infant Pain Scale (NIPS), Neonatal Facial Coding System (NFCS), Premature Infant Pain Profile (PIPP) scores were evaluated before the injection, during the injection, and at 2 minutes after the injection. Pain scores were compared among the 4 groups. Results: Maternal and neonatal characteristics were similar among the 4 groups. 25% dextrose treatment led to lower NIPS pain scores during injection (6.4±0.9 vs. 5.5±1.7, P=0.01) and after injection (1.6±2.0 vs. 0.6±0.9, P=0.01) and NFCS pain scores after injection (1.5±2.3 vs. 0.7±0.8, P=0.04) than control group. The number of neonates who feel the pain (indication of scores: NIPS≥4, NFCS≥3) decreased (9 (23.1%) vs. 0 (0%), P=0.04 via NIPS, 7 (17.9%) vs. 0 (0%), P=0.02 via NFCS). However, all treatment groups did not decreased PIPP scores, compared with the control group. Conclusion: Oral 25% dextrose is effective than distilled water or using pacifier with or without 25% dextrose in reducing pain during intramuscular injection of hepatitis B vaccinations. Further study based on this preliminary study need about nonpharmacologic management of pain in newborns.